Project Summary: Type II CRISPR/Cas9 systems are revolutionizing biomedical science. These programmable nucleases facilitate the creation of a double strand break at a specific location within a genome, which promotes targeted gene disruption or gene editing through homologous repair with an exogenously supplied donor DNA. While existing Cas9 systems are powerful, their promiscuity presents a barrier to their implementation in gene therapy applications, where undesired collateral damage to the treated genome must be minimized or, ideally, eliminated. Consequently, further development of this nuclease platform for the selective recognition and cleavage of a desired target sequence (and only that sequence) is warranted. To achieve the ultimate goal of single-site nuclease precision within the human genome, we propose to develop a chimeric fusion between Cas9 and a programmable DNA-binding domain (pDBD). We have established and validated a working prototype that has improved precision, greater activity, and a broader sequence targeting range than the standard Cas9 system. In this proposal, we outline experiments to use appended pDBDs to improve precision of three representative, validated Cas9 orthologs: S. pyogenes and S. aureus Cas9 (SpCas9 & SaCas9; representative Type II-A) and N. meningitidis Cas9 (NmCas9; representative Type II-C). These systems will be applied to the therapeutic gene correction of chronic granulomatous disease (CGD). In Aim 1, we will optimize the characteristics of our established SpCas9-pDBD fusions to create a chimeric system that requires an additional stage of licensing for target cleavage and incorporates exogenous regulation through a drug-dependent dimerization system. In Aim 2, we will extend the advantages of Cas9-pDBD fusions into the more compact NmCas9 and SaCas9, and identify the similarities and differences in essential design principles between Type II-A and Type II-C Cas9-pDBD fusions. In Aim 3, we will apply our Cas9-pDBD system to the precise and efficient correction in hematopoietic stem cells of X-linked defects that are associated with CGD to establish a gene correction-based autologous stem cell therapy for this devastating disease. Ultimately, the proposed research promises to yield genome-editing enzymes that exhibit the specificity required for safe, effective application in clinical gene therapy and stem cell engineering, which we will demonstrate by creating a cell-based gene therapy for CGD.